DNA-PKcs and PIDD interaction in DNA damage response

DNA 损伤反应中 DNA-PKcs 和 PIDD 相互作用

• 批准号: 10228547
• 负责人: Benjamin Ping-Chi Chen
• 金额: $ 37.06万
• 依托单位: UT SOUTHWESTERN MEDICAL CENTER
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-09-01 至 2024-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10228547
• 关键词: Affect; Apoptosis; Attenuated; B-Cell Lymphomas; CASP2 gene; Cancer Model; Catalytic Domain; Cell Culture Techniques; Cell Death; Cell Differentiation process; Cell Line; Cell Proliferation; Cell Survival; Cells; Complex; DNA; DNA Damage; DNA Double Strand Break; DNA Repair; DNA Repair Pathway; DNA lesion; DNA replication fork; DNA-PKcs; DNA-dependent protein kinase; Death Domain; Development; Double Strand Break Repair; G22P1 gene; Genome; Holoenzymes; In Vitro; Incidence; Inflammation; Investigation; Ionizing radiation; Liver; Malignant Neoplasms; Malignant neoplasm of liver; Mediating; Mediator of activation protein; Modeling; Mus; Mutation; Nonhomologous DNA End Joining; Normal Cell; Oncogene Activation; Oncogenes; Pathway interactions; Phosphorylation; Phosphotransferases; Play; Primary carcinoma of the liver cells; Process; Proteins; Radiation Tolerance; Radiation exposure; Reagent; Recovery; Regulation; Reporting; Resistance; Role; Signal Pathway; Steatohepatitis; TP53 gene; Testing; Tissues; UV carcinogenesis; Ultraviolet Rays; Work; ataxia telangiectasia mutated protein; base; biological adaptation to stress; cancer cell; cancer prevention; carcinogenesis; genome integrity; in vivo; male; mouse model; mutant; novel; preservation; prevent; recruit; regenerative; repaired; replication stress; response; senescence; tumorigenesis; ultraviolet irradiation

Project Summary/Abstract The DNA dependent protein kinase catalytic subunit (DNA-PKcs) is a key regulator of the canonical non- homologous end-joining (NHEJ) pathway for repair of DNA double strand breaks (DSBs) and resistance to ionizing radiation (IR). DNA-PKcs is recruited by the Ku70/80 heterodimer to the DSB ends to form the DNA-PK holoenzyme to initiate NHEJ mechanism. DNA-PKcs also plays an important role in cellular resistance to replication stress. It coordinates with and is rapidly phosphorylated by the ATR (ataxia telangiectasia mutated and Rad3 related) kinase at stalled replication forks upon UV, although the mechanism is not well understood. Our recent work has focused on identifying the player(s) required for the recruitment of DNA-PKcs to stalled replication forks. We have identified that PIDD (p53-induced protein with a death domain), a known apoptosis mediator for assembling the PIDDosome complex and Caspase-2 activation, is required for DNA-PKcs recruitment to stalled replication forks and its association with ATR. Disrupting the interaction between DNA- PKcs and PIDD not only compromised ATR dependent DNA-PKcs phosphorylation at the Thr2609 cluster but also attenuated the ATR signaling pathway and intra-S checkpoint. To assist our investigation, we have created cell lines and a mouse model expressing a DNA-PKcsPL mutant protein unable to interact with PIDD. Our results showed that DNA-PKcsPL cells were highly sensitive to both UV and IR. Based on these preliminary findings, we hypothesize that PIDD, but not the Ku70/80 heterodimer, mediates DNA-PKcs recruitment to stalled replication forks and promotes its association with the ATR pathway. We also hypothesize that PIDD facilitates DNA-PKcs kinase activation and DSB repair. Finally, we hypothesize that the interaction of DNA-PKcs with PIDD will affect cell death regulation and cancer development. In this project, we will determine the coordination between DNA- PKcs, PIDD, and ATR in the cellular response to replication stress. Our specific aims are: 1) To test the hypothesis that PIDD modulates DNA-PKcs kinase activation at stalled replication forks and at IR-induced DNA lesions that are not bound by Ku70/80, 2) To test the hypothesis that PIDD is required for DNA-PKcs to properly function at stalled replication forks upon UV irradiation, and 3) To test the hypothesis that the DNA-PKcs-PIDD association affects cell fate determination upon DNA damage and cancer development.

项目总结/摘要 DNA依赖性蛋白激酶催化亚基（DNA-PKcs）是典型非蛋白激酶活性的关键调节因子。 同源末端连接（NHEJ）途径修复DNA双链断裂（DSB）和抗 电离辐射（IR）。DNA-PKcs被Ku 70/80异二聚体募集到DSB末端以形成DNA-PK 全酶启动NHEJ机制。DNA-PKcs还在细胞对肿瘤的抗性中起重要作用。 复制应力它与ATR（共济失调毛细血管扩张症突变型）协调并被ATR快速磷酸化。 和Rad 3相关）激酶，尽管其机制还不清楚。 我们最近的工作集中在确定DNA-PKcs的招募所需的参与者， 复制分叉。我们已经鉴定了PIDD（p53诱导的死亡结构域蛋白），一种已知的细胞凋亡蛋白， 组装PIDDosome复合物和Caspase-2激活的介体是DNA-PKcs所需的 招募停滞的复制叉及其与ATR的关联。破坏DNA之间的相互作用- PKcs和PIDD不仅损害了Thr 2609簇的ATR依赖性DNA-PKcs磷酸化， 也减弱ATR信号通路和intra-S检查点。为了协助我们的调查， 细胞系和表达不能与PIDD相互作用的DNA-PKcsPL突变蛋白的小鼠模型。我们的结果 结果表明，DNA-PKcsPL细胞对紫外线和红外线都高度敏感。基于这些初步发现，我们 假设PIDD而不是Ku 70/80异二聚体介导DNA-PKcs募集到停滞复制 分叉并促进其与ATR途径的关联。我们还假设PIDD促进DNA-PKcs 激酶活化和DSB修复。最后，我们假设DNA-PKcs与PIDD的相互作用将影响PIDD的表达。 细胞死亡调节和癌症发展。在这个项目中，我们将确定DNA之间的协调- PKcs、PIDD和ATR在细胞对复制应激反应中的作用我们的具体目标是：1）测试 假设PIDD在停滞的复制叉和IR诱导的DNA上调节DNA-PKcs激酶活化 2）为了检验PIDD是DNA-PKcs正常表达所必需的假设， 3）为了验证DNA-PKcs-PIDD在紫外线照射下在停滞的复制叉中起作用的假设， 相关性影响DNA损伤和癌症发展后的细胞命运决定。